WO1991014706A2

WO1991014706A2 - Lectin affinity membranes and methods for using them

Info

Publication number: WO1991014706A2
Application number: PCT/US1991/001578
Authority: WO
Inventors: Oscar Dile Holton; Yves Fouron
Original assignee: Sepracor, Inc.
Priority date: 1990-03-19
Filing date: 1991-03-08
Publication date: 1991-10-03
Also published as: WO1991014706A3

Abstract

This invention relates to lectin affinity membranes and methods for carrying out affinity separations of glycosylated proteins using them. The method utilizes a membrane-bound lectin ligand first to capture and separate a glycosylated protein from a fluid mixture and subsequently to release said protein in purified form. The present affinity membrane methods recover high-purity glycosylated proteins. The invention has particular relevance to the recovery and purification of glycosylated proteins from mixtures of biological origin.

Description

LECTIN AFFINITY MEMBRANES

AND METHODS FOR USING THEM

Description Background of the Invention

Affinity Separations

Of the separation technologies available today, those based on affinity interactions are ever more popular, particularly at the laboratory scale. Affinity separation has become the preferred method for purifying proteins and other biomolecules from complex,

biologically derived fluids. Affinity Chromatography and Biological Recognition, I.M. Chaiken, M. Wilchek and I. Parikh (eds.), Academic Press, New York, 1983; Hill, E.A. and M.D. Hirtenstein, "Affinity chromatography: its application to industrial scale processes", Advances in Biotechnological Processes, Alan R. Liss, Inc., New York,

1983). The key to the method's attractiveness is its unequaled degree of selectivity.

Affinity separations, as they are conventionally practiced, typically involve a number of sequential steps. First, a solution containing a component to be separated from the solution (a component of interest) is passed through a column containing a highly specific ligand which will reversibly bind the compound of

interest immobilized on a support, usually

high-surface-area beads or particles. As the fluid passes through the column in this loading step, the desired component binds selectively and reversibly to the immobilized ligand, while most impurities pass unhindered. Residual impurities are removed by flushing the column with an appropriate buffer solution in a subsequent washing step. The component, now purified but still bound to the immobilized ligand, is then recovered by passing an eluent solution through the column that has the effect of disrupting the ligand-to-ligate binding interaction. Generally, the pH, concentration of a salt, or some other chemical characteristic of this eluent solution is altered significantly from the corresponding values of the loading and wash solutions, and it is this change that is responsible for weakening the affinity interaction and thereby causing desorption and elution of the ligate molecule.

Many types of molecules can serve as ligands, including antibodies, antigens, enzyme inhibitors, lectins, isolated receptors, and more recently, cloned receptors. Bailon, P. et al., Bio/Technology 5:1195

(1987). In contrast, however, the choice of materials to support the ligand has been somewhat limited. Agarose gel beads (e.g., 50 to 150 microns diameter) have

traditionally received the most attention as affinity ligand supports, particularly on the laboratory scale. Within recent years, cross-linked and accordingly more rigid versions of these and other polysaccharide-based gel beads have been developed and introduced, as have various microporous support particles based on synthetic polymer compositions. These polymeric support materials are now complemented by various inorganic materials. For example, porous silica packed in high-pressure columns is used to perform affinity separations in an HPLC-like process. Typical pore diameters in the silica support range from about 200 to about 1000 Angstroms, whereas silica particle diameters are generally in the range of about 5 to 25 microns. Affinity Membranes

Affinity separation processes for the recovery and purification of proteins are conventionally carried out using sorbent beads or particles packed in columns, as discussed above. The adsorption process is carried out in a cyclical fashion comprising four steps:

1. Load : A solution of target component in a

mixture is made to pass through a packed column; target component ("ligate") is recognized and captured by the immob il iz ed sorbent ("ligand"), while most contamnnants pass through. 2. Wash: A wash solution is passed through the column to flush out contaminants present in the column void volume as well as to displace non-specifically bound contaminating sub- stances.

3. Elute: An eluent solution is passed through the column to disrupt the affinity binding between immobilized ligand and reversibly bound ligate, causing elution of the latter from the column in a purified condition.

4. Regenerate: A regeneration solution is passed through the column in order to return it to conditions (e.g., pH and/or ionic strength) that favor ligand/ligate binding. Despite the high selectivity that affinity processes provide, however, their application on the process scale has been hampered by the inability of affinity columns to handle high flowrates at reasonable ligand utilization efficiencies.

Affinity membrane devices are based on microporous membranes, preferably hollow fibers activated by covalent attachment of affinity ligands to the interior surfaces of the membrane's pore walls. In operation, feed

solution is made to flow through the membrane from one of its surfaces to the other, during which process the target molecule is recognized and captured by the

immobilized ligand which it encounters, leaving the filtrate devoid of ligate. Like columns, these affinity membranes can be operated in a cyclic affinity adsorption process to produce high-purity protein in a single step. However, unlike columns based on particulate affinity ligand supports, affinity membranes are not hampered by the serious pressure drop and mass transfer limitations from which columns suffer. As a result, affinity membranes are capable of operating at higher volumetric throughputs and ligand utilization efficiencies than are columns.

Both polymeric and inorganic affinity support particles suffer from hydrodynamic or pressure drop limitations when used in columns. With the former (e.g., soft agarose gel beads), particle compressibility is a problem, inasmuch as attempts to increase flowrate through a column packed with agarose are normally met by increased pressure drops. This leads in turn to further compression of the particles and reduced bed

permeability. Clonis, Y.D., Bio/Technology 5 : 1290 ( 1987). It is only by resorting to very shallow but largediameter packed columns (i.e., columns with a relatively large ratio of bed diameter to depth) that practical volumetric throughputs can be obtained. Alternatively, one can resort to more rigid particles (e.g., silica, controlled-pore glass), but here the small size of the support particles limits volumetric throughput unless high operating pressure are employed.

In contrast, affinity membranes with adsorptive pore walls provide extremely short fluid-flow path lengths in comparison to the superficial area provided for flow. This unique geometry of affinity membranes thus leads to very high fluid throughputs per unit of applied pressure difference as compared to affinity columns.

Another important consideration in evaluating the merits of membranes vs. columns as affinity ligand supports is the matter of their relative mass transfer efficiency. Efficient capture of a target protein in an affinity column requires that the characteristic time for diffusion of protein to the immobilized ligand be short as compared to the residence time of fluid in the column. If this condition is not met, premature breakthrough is encountered and the "dynamic" sorption capacity of the bed will not approach its "static" or equilibrium

capacity.

A characteristic diffusion time for the encounter between diffusing ligate and immobilized ligand can be defined as the ratio of the square of a characteristic diffusion distance to the diffusivity of the ligate molecule. The required residence time of fluid in the affinity device during the loading step will increase in proportion to this characteristic diffusion time. Thus, in order to keep the characteristic distance for ligate diffusion into the support as small as possible (and thereby to maximize device throughput during loading), it is necessary to use support particles that are as small as practical (e.g., fine silica or synthetic polymeric particles). However, doing so tends to aggravate the above-mentioned pressure drop problem, forcing one away from low-pressure operation towards a high-pressure liquid chromatography process.

In contrast, affinity membranes obviate the need to work with small (e.g., micron-sized) particles in order to minimize diffusion distances and diffusion times.

Where protein-containing solutions are pumped across affinity membranes, the characteristic distance across which ligate must diffuse in order to meet membrane-bound ligand is of the order of a quarter of the pore diameter; typically, this diffusion distance is only a fraction of a micron. Because diffusion time varies with the square of diffusion distance, the impact of the reduction in diffusion distance afforded by affinity membranes on improved mass transfer efficiency and volumetric productivity is dramatic. These and other aspects of affinity membrane performance have been discussed by S. Brandt et al., Bio/Technology 6:152 (1988) and in co-pending U.S. Applications Serial No. 07/265,061 and No. 07/428,263, referred to above.

The Use of Lectins as Li gands

Lectins are generally identified by their ability to agglutinate red blood cells from a broad range of

species. Many lectins have also been described that will only bind to blood cells after enzyme treatment (i.e., pronase, trypsin, or neuraminidase). In order for agglutination to occur, a lectin must possess at least two carbohydrate binding sites. The ability of the lectins to agglutinate red blood cells is the basis for their being defined as proteins (or glycoproteins) of non-immune origin that agglutinate cells or precipitate complex carbohydrates. This definition therefore

excludes glycosidases which are specific for certain carbohydrate structures but which do not agglutinate cells. In general the majority of lectins may be inhibited by monosaccharides, although di- and trisaccharides are more potent inhibitors for many lectins.

Lectins have proven to be extremely versatile tools in the field of carbohydrate research. One of the areas of active research has been in immobilization of lectins on a support matrix and using this lectin matrix to purify glycoproteins. Lectins that have been covalently linked to a support matrix have been used successfully for identification of pathogenic bacteria by latex agglutination, for cell fractionation, and for affinity chromatography of abroad range of glycoproteins and glycopeptides. Young and Leon, Biochim. Biophys. Acta.,

365:418 (1974); Kornfeld et al., J. Biol. Chem. 246:6581 (1971); Kleine et al., Molecular Immunol. 16:421 (1979). Immobilized lectins for affinity chromatography have an advantage over many other purification techniques since the protein to be purified is generally not subjected to harsh or denaturing conditions.

Lentil lectin is a hemagglutinating lectin isolated from the common lentil Lens culinaris (also known as Lens esculenta) and shows a specific binding affinity towards α-D-glucose and α-D-mannose residues. Although lentil lectin was described by Landsteiner and Raubitschek

(Zentralbl. Bakteriol. Parasitenkd. Infektion Hyg.,

45:660 (1908)), it was not isolated and fully

characterized until sixty years later. Entlicher et al., Experientia, 25:17 (1969); Howard and Sage, Biochemistry, 8: 2436 (1969); Ticha et al., Biochem Biophys. Acta.,

222:282 (1970).

Lentil lectin has two carbohydrate binding sites per molecule of molecular weight 52,000. Lentil lectin activity is associated with two closely related isolectins which are known as LcH A and LcH B. Each isolectin is a tetramer containing two different subunits of MW = 6,000 and 18,000. Both isolectins are primarily specific for alpha-mannose residues. The presence of an alpha-L-fucose residue attached to the asparagine-linked GlcNAc residue of complex oligosaccharides contributes to a stronger binding than that seen with other similar lectins (i.e., Concanavalin A or Con-A).

Initial utilization of lentil lectin in purification schemes demonstrated that glycoproteins like α₂-macroglobulin, IgM, Gc-globulin and β₂ -glycoprotein would bind to the lectin. Other glycoproteins such as transferrin, ceruloplasmin, haemopexin, haptoglobin and α₁-acid glycoprotein did not bind to lentil lectin. Young et al., J. Biol. Chem. 246:1596 (1971). A glycopeptide from ovalbumin known to bind to other lectins had little or no binding affinity for lentil lectin.

Immobilized lentil lectin is a generally applicable group-specific adsorbent for the purification of glycoproteins by affinity chromatography. Lentil lectin immobilized on Sepharose™ has, for example, been used to purify rat brain acetylcholinesterase and for fractionating viral proteins from influenza, mouse mammary tumor and Sendai viruses. Hayman et al., FEBS Lett., 29:185 (1975).

Lentil lectin immobilized on Sepharose™ has been widely used for affinity chromatography of membrane proteins, since the carbohydrate-binding activity of the lectin is retained in the presence of detergents at concentrations (about 1%) used routinely for solubilizing membrane proteins. Glycoproteins from the plasma membranes of normal and virus-transformed fibroblasts can be isolated this way. Pearls tein, Exp. Cell . Res ., 109:95 (1977). The glycoproteins purified in this manner represent only approximately 5% of the total plasma membrane proteins. Immobilized lentil lectin has been found to be especially useful for the isolation of membrane glycoproteins from lymphoid tissue with overall recovery of certain glycoproteins as high as 95%. Lentil lectin has also been reported to be the most suitable immobilized lectin for the isolation of mouse H-2 and human HLA antigens. Kvist et al., Biochemistry, 16:4415 (1977); Dawson et al., J. Immunol., 112:1190 (1974).

The limited literature descriptions of purification of IgM using immobilized lentil lectin generally are based on individual experiments in a described condition. These reports describe methods that require immobilizing lentil lectin and lectins from other sources on a

Sepharose™ support. Kornfeld et aI., J. Biol. Chem.,

256:6633 (1981); Pharmacia Fine Chemicals, "Lentil

Lectin, Lentil Lectin-Sepharose 4B, For Cell Surface Studies And Affinity Chromatography" (1978); Harris and Robson, Vox Sang, 8:348 (1963); Takacs and Stachelin,

Immunol. Methods, Vol. II, pp. 27-56 (1981); Hayzer and Jaton, Methods in Enzymology, Vol. 116, pp. 26-36 (1985). The sample containing the IgM, usually serum or ascites fluid, is then diluted approximately 1:10-1:100 and allowed to flow over the lectin Sepharose™ support.

Following a wash step to remove excess contaminating proteins, the IgM glycoproteins remaining attached to the support are eluted using a competing carbohydrate

molecule, usually methyl-α-D-mannopyranoside or methyl- α-D-glucopyranoside. The concentration of competing carbohydrate ranges between 0.2 M and 0.4 M. The lentil lectin support is then regenerated for processing another glycoprotein solution. Exact definition of the important parameters for good purification of IgM by lentil lectin has not been reported.

The use of membranes as affinity ligand supports has been shown to provide certain advantages in affinity, separations (e.g., high volumetric productivity,

efficient use of ligand, and scaleability) that are related to the unique aspect ratio of membranes relative to conventional columns and to their superior mass transfer characteristics. Summary of the Invention

The present invention relates to unique lectin affinity membranes, methods for making the membranes and methods for carrying out affinity membrane separations of glycosylated proteins using the membranes. The methods are carried out under conditions which are optimized for recovering pure, glycosylated proteins and maintaining affinity ligand activity.

The present membranes are microporous membranes having lectin ligands immobilized thereon. The lectins are attached to the membranes using unique coupling chemistries. In one embodiment, the microporous membrane is activated using an activating substance such as

2-fluoro-1-methyl-pyridinium p-toluene-sulfonate (FMP) or dialdehydes (such as glutaraldehyde) and other immobilization chemistries known in the art, followed by incubation with a solution of the lectin to be immobilized. The resulting membranes have lectins covalently attached to the membrane pore wall surfaces. A method of separating glycosylated proteins using the present membranes is also the subject of the present invention. The use of the present affinity membranes allows efficient separation of glycosylated proteins using lower concentrations of sugar eluent than is necessary to effect separation of the same proteins by conventional (i.e., non-membrane) chromatography methods. The present method relies upon an eluent mixture

containing a low concentration of sugar and a salt. In another embodiment of the present method, a sugar-less, salt only eluent is used to elute the protein. Both eluents provide high levels of purity and high yields of the protein. In the present method, a glycosylated protein associated with a lectin ligand, such as lentil lectin, immobilized on an affinity membrane is contacted with an eluent having a low sugar concentration, e.g., about 0.2 M, or less, and a salt concentration typically of about 0.5 M up to about 1.0 M. Substantially all of the protein elutes under these conditions in a very pure form. The term "associated with" as used herein generally means that the ligate is reversibly bound to the ligand. Due to the properties obtainable in affinity separations with microporous membranes much milder elution conditions can be used. For example, an eluent which is free of sugar or one having a sugar concentration of from about 0.05 M to about 0.4 M is used in the membrane affinity system, wherein an eluent having a sugar concentration of from about 0.4 M to about 0.6 M would be necessary to elute the same protein in about the same yield and purity from a conventional non-membrane support matrix. The present method is based on the discovery that it is possible to exploit the unique characteristics of lectin-based affinity membranes for the purpose of improving the purity of active product and useful lectin life. In addition, the present method utilizing

relatively small amounts of immobilized lectin ligand, and eluents having lower sugar concentrations improves the cost-effectiveness and therefore the commercial utility of the method. The present method is a membrane- based affinity system which provides good yields of active glycosylated proteins in affinity separation processes. The present lectin-bound affinity membranes exhibit better ligand utilization than is available using non-membrane matrices. Use of the present membranes allows significantly less ligand to be used than

conventional affinity techniques, which reduces both the cost and the problems associated with ligand leaching which can occur in conventional (non-membrane) systems. Brief Description of the Figures

Figures 1A and 1B are chromatograms illustrating the results of a GPC analysis of A) mouse ascites feed fluid, diluted to an IgM concentration of 0.08 mg/ml; and B) IgM purified from the mouse ascites by a lectin affinity membrane process.

Figures 2A and 2B are chromatograms showing the results of a GPC analysis of A) a clarified, serum-free cell culture supernatant from CHO cells containing about 50 μg/ml recombinant MCSF; and B) the recombinant MCSF purified from the cell culture fluid by a lectin affinity membrane process.

Figures 3A, 3B and 3C are chromatograms illustrating the results of a GPC analysis of a two step IgM

purification consisting of: A) a mouse ascites feed solution containing IgM; B) the IgM purified by a lectin affinity membrane process; and C) the IgM of B further purified by Ion exchange chromatography.

Figures 4A and 4B are chromatograms illustrating the purity of IgM purified using A) a lentil lectin affinity membrane; and B) a lentil lectin-Sepharose matrix.

Figure 5 is a schematic illustration of a representative affinity separation system. Detailed Description of the Invention

The present invention relates to affinity membranes having lectin ligands covalently bound to the membranes, methods of making the lectin-bound membranes and methods of separating glycosylated proteins using the lectin- bound membranes. In this section, the basic elements of an affinity membrane system are described, along with a recitation of the various steps involved in its use in purifying a glycosylated protein from a complex biological mixture according to the method of the present invention.

The affinity membranes used in the present invention are microporous membranes. Microporous membranes suitable for affinity chromatography and methods of making them are described in detail in co-pending U.S. patent application Serial No. 07/258,406, filed October 17, 1988, the teachings of which are incorporated herein by reference. As stated In detail therein, microporous membranes are generally produced by casting or extruding polymers, such as polysulfones, polyethersulfones, polyimides, poly(arylene oxides), polyarylene sulfides, pblyquinoxalines, polysilanes, polysiloxanes, poly- urethanes, poly(etheretherketones), polycarbonates, polyesters, poly(vinylhalides), poly(vinylidene poly- halides) and copolymers and/or blends of the above. The polymer surface can be treated to alter the properties of the membrane. A linker molecule which is capable of serving as a covalent bridge is introduced, which allows a ligand or macromolecule to be attached which alters the surface or interfacial properties of the membrane. In addition to the linker molecule, an activating reagent is generally needed to covalently bind the ligand of choice to the polymer. Activating reagents render selected functional groups of macromolecules already bound to the polymer surface more reactive towards the functional groups of the added ligand as described In the above- referenced patent application, U.S. Serial No.

07/258,406.

Membranes useful in the present invention are produced by activating a microporous membrane having hydroxyl functionality by employing an activating reagent which renders the membrane surface receptive for covalently binding lectins. Activating reagents useful for this purpose include, but are not limited to, 2-fluoro- 1-methylpyridinium-p-toluenesulfonate (FMP), and

dialdehydes, such as glutaraldehyde. Both of these reagents facilitate the coupling reaction with lectins by introducing a superior leaving group. In one embodiment of the invention, a hydroxyl-end-group-containing poly- ethersulfone microporous membrane is modified by applying a hydroxyl-containing coating, such as hydroxyethyl cellulose, to the membrane, thus amplifying the number of hydroxyl groups available on the membrane surface, or with polyethyleneimine, thus providing amine functionality on the molecule. The hydroxyl functional membrane is then treated with FMP, and the amino - functional membrane with glutaraldehyde to activate the membrane surface. The activated membrane is then exposed to a solution of the lectin to be bound under conditions sufficient for covalent attachment of the lectin to the membrane to occur.

Any lectin can be used as the ligand in the present affinity membranes and methods of making them using the

FMP and aldehyde linking chemistries. The term "lectins" as used herein refers to a class of proteins which have the ability to agglutinate erythrocytes and other types of cells. Lectins are generally defined as sugar-binding proteins or glycoproteins of non-immune origin which agglutinate cells and/or precipitate glycoconjugates. Lectins which are particularly useful in the present invention are plant lectins, which include concanavalin-A (Con-A) lectin, wheat germ agglutinin (WGA) and lentil lectin.

The lectin-functional affinity membranes produced by the method have several advantages over lectins bound to a non-membrane support matrix. The present lectin-bound membranes have a higher "static" or "equilibrium"

capacity than non-membrane support-bound lectins, and exhibit better ligand utilization. The term "static capacity" as used herein refers to the amount of ligate that can be bound to a given volume of the membrane surface where the system is in equilibrium, i.e., when the concentration of ligate in the solution being passed over the membrane is in such great excess that all possible binding sides are occupied and the bound ligate is in equilibrium with the nonbound ligate. The static capacity of the membrane matrix is about three (3X) times greater than Sepharose™, for example, as shown in Table I:

*Both support matrices have immobilized lentil lectin ligands

**CNBr = cyanogen bromide

Ligand utilization refers to the amount of ligand necessary to purify (i.e., to bind and release) a given quantity of ligate per unit time. The present membranes turn over ligand at a significantly faster rate than non-membrane support matrices, contributing to better ligand utilization.

Methods of separating and/or purifying glycosylated proteins using the lectin affinity membranes are also the subject of the present invention. The method is

particularly useful for purifying glycosylated proteins such as macrophage colony stimulating factor (MCSF) and immunoglobulin M (IgM).

As mentioned above, there are four phases in the membrane affinity purification process, namely, loading, washing, elution, and regeneration. The process is described in detail in copending applications Serial No. 07/428,263, Serial No. 07/265,061 and 07/258,406, the teachings of all of which are incorporated herein by reference. The process is briefly described below. Affinity Membrane Purification Method and Apparatus Biological products are captured by an affinity membrane module through formation of reversible complexes between ligand molecules immobilized on the pore wall surfaces of the membrane and biomolecules in solution. Particularly useful as affinity membranes are microporous hollow-fiber membranes, e.g., porous membranes comprised of a polysulfone-containing substrate material coated with a hydrophilic material and subsequently activated for covalent attachment of a ligand. Prior copending U.S. Application Serial No. 07/258,406 filed October 17, 1988 describes the preparation of a suitable microporous hollow-fiber membrane, along with various procedures for coating, activating, and linking various ligands to it. For capture and purification of glycosylated proteins, lectin ligands can be attached to the interior membrane surfaces using, for example, FMP or glutaraldehyde linking chemistries, as well as other chemistries that are well known in the art of affinity chromatography. A method for immobilizing lentil lectin on a membrane using glutaraldehyde linking chemistry is described in detail in Example 5. A schematic illustration of an apparatus which is useful for affinity separations is shown in Figure 5.

Without wishing to be limited as to module size, typical affinity membrane modules range in size from 1.5 mL to 1L total volume (i.e., total device size, about one-third of which is occupied by the porous matrix that comprises the hollow-fiber affinity membrane walls). The 1.5 mL and 30 mL modules are particularly preferred for use in conjunction with the automated apparatus described herein, whereas 150 mL and 1L modules are more suited to process-scale applications.

The feed to the affinity membrane process may consist of practically any glycosylated protein- containing fluid; examples include mammalian cell culture supernatants, ascites fluids, fermentation broths, or blood and blood plasma. This fluid will generally be at near-neutral pH and otherwise physiological conditions. Pretreatment or clarification of the fluid by various standard methods (e.g., microfiltration) may be required in order to prevent excessive membrane fouling.

It should be noted that the particular compositions recited above are meant solely to serve as examples; they are not limiting as to the practice of the process of the present invention.

Loading

To begin the purification cycle, the sample containing glycosylated protein is loaded onto the membrane-bound lectin ligand while the system is

typically at a pH in the range of about 7 to about 8

(e.g., about pH 7.4). Sample fluid containing glycosy- lated protein is circulated from the feed reservoir through the affinity module at a preset rate. As the protein passes through the affinity membrane, it binds to the immobilized lectin ligand. In the module, the fluid divides into two paths, referred to as the shell and lumen paths (as shown in Figure 5). The lumen path is followed by that portion of the feed fluid that does not pass through the membrane but that flows out the affinity module through the lumen outlet still carrying its original concentration of glycosylated protein. This undepleted fluid returns to a feed reservoir for

recycling and eventual recovery of the protein contained within it. The shell path is used to describe the path of the fluid entering the affinity module which is drawn through the hollow-fiber membrane wall by the filtrate pump. This fluid loses its glycosylated protein to the lectin ligand, so that it emerges from the shell-side surface of the membrane devoid of much or all of its glycosylated protein. This filtrate is now pumped from the shell outlet of the affinity module to the waste reservoir at the preset rate.

During this loading phase, the filtrate flowrate is always less than the feed flowrate. Both UV detectors operate in the loading phase, providing a continuous record of the absorbance (and hence total protein concentration) of both feed and filtrate streams as they emerge from the module.

Washing

Unbound glycosylated protein remaining in the affinity module may be washed out with a neutral

physiological buffer, e.g., Tris or phosphate buffered saline (PBS) typically at about pH 7.4, in a three-part process. In the shell wash the shell (external) side of the hollow fibers is washed to remove any remaining protein-depleted sample fluid. The buffer is drawn by the filtrate pump into the shell inlet and out the shell outlet, subsequently to be routed to the waste reservoir.

The operation of the affinity purification system during the lumen washes is as described below and shown in Figure 5. In these steps, the lumen (internal) side of the hollow fibers is washed free of cell culture fluid. The wash buffer is drawn by the filtrate pump into the shell inlet, through the membrane, out the lumen outlet, and then past the feed UV detector. In the first lumen wash, residual cell culture (or other) fluid returns to the feed reservoir for eventual capture of the glycosylated protein that it contains.

Elution

The glycosylated protein is released from the lectin ligand in a series of elution steps. The protein product is eluted during each of the steps of this phase, but generally it is collected only during the main elution step.

In the elution step of the present process, the eluent which is used to detach the glycosylated protein from the lectin ligand is an aqueous solution which contains a sugar having a concentration of from about 0.05 M to about 0.4 M, and a salt having a concentration of from about 0.3 M to about 1.0 M. Sugars which are useful in the present method include sugars containing _-D-glucose or α-D-manriose residues. Sugars which are particularly useful, for example, are methyl-α-dD-gluco- pyranoside and methyl-α-D-raannopyranoside. Salts which are useful as eluents in the present method include alkali metal salts of halogens, for example. A salt which is particularly useful is sodium chloride (NaCl). In one embodiment of the present method, salt alone, without sugar, is used as the eluent. Salt solutions which can be used are those having the same concentration range as set out above for the sugar/salt combinations.

In the shell flush step, eluent drawn by the filtrate pump flows in the shell inlet, flushes out the wash buffer, and then flows through the shell outlet to the waste reservoir.

During pre-elution, release of the glycosylated protein begins as the eluent flows into the affinity module through the shell inlet, through the membrane, and out the lumen outlet. Fluid then flows to the waste reservoir, passing the feed UV detector along the way.

A subsequent post-elution step, conducted after the main elution step discussed below, is similar, with eluent here being directed to the waste reservoir as release of the protein falls from peak levels. The fluid follows the same path as in the pre-elution step to the waste reservoir.

During the main elution step, when a 1.5 mL affinity membrane module is In place, approximately 8-9 mL of product-bearing fluid is released from the ligand; during this step, the ligate release rate reaches its maximum value. Eluent follows the same path as in the previous step, but it now flows to the product reservoir for collection rather than to the waste reservoir for

disposal.

Regeneration

The affinity purification system is restored to original starting conditions in this step by flushing with a) a regeneration buffer and b) a equilibration buffer. The regeneration buffer, such as PBS or Sodium Borate, or Sodium Acetate, 0.05% Tween 20, pH 7.4, containing ImM MnCl₂ and ImM CaCl₂, in a two part

sequence, is flushed to the waste reservoir.

In the shell regeneration step, the shell-side volume in the follow-fiber affinity membrane module is restored to neutral pH as the regeneration buffer is drawn by the filtrate pump into the shell inlet and out the shell outlet.

In the subsequent lumen flush regeneration step, the lumenal volume of the hollow-fiber module is restored to neutrality. The regeneration buffer is drawn by a filtrate pump into the shell inlet, through the membrane, and out the lumen outlet, thus preparing the affinity purification system to begin another purification cycle.

Following the regeneration buffer steps the affinity system is flushed with an equilibration buffer, such as 20 mM Tris, 0.05% Tween 20, pH 7.4, in a two step

sequence. All material is flushed to the waste

reservoir.

Examples of protein purification by this affinity membrane method and apparatus are set out in detail in prior copending U.S. Application Serial Nos. 07/265,061 and 07/428,263 (see in particular Sections 6.1-6.6 and 6.11 therein). Release of Lectin-Bound Glycosylated Protein In exploring the operating limits of the affinity membrane purification apparatus discussed in the preceding section, it was discovered that it is possible to displace lectin-bound glycosylated protein at

substantially lower sugar concentrations than those which are typically prescribed with more conventional gel- and particulate-type affinity media used in packed columns and stirred tanks. This unexpected ability to elute glycosylated protein ligates from immobilized lectin affinity membranes at significantly lower sugar concentrations than heretofore realized has an important benefit, namely, that smaller quantities of oftenexpensive sugar eluting solutes may be employed, with a corresponding improvement in process economics. Additionally, in conventional systems such as lectin- Sepharose™, the rate of leaching of lentil lectin from the support matrix has been shown to increase with higher sugar concentrations. The use of lower sugar concentrations to elute lectin-based affinity membranes may lead to increased immobilized ligand life.

For example, elution of MCSF from particulate or bead-type chromatographic media activated with lentil lectin is frequently effected by contacting the matrix with a concent-rated sugar solution, typically, 0.4 M methyl-α-D-glucopyranoside. The same or similar eluents can also be used in affinity membrane purification, as has been described above, but large amounts of the sugar eluting solute can be quite expensive. The need clearly exists for affinity separation methods that permit elution of glycosylated proteins from lentil lectin affinity supports with reduced amounts of sugar eluting solutes.

In addition, the purity of the glycosylated proteins, particularly IgM, obtainable from the immobilized lectin affinity membrane process is much higher than conventionally obtained, thereby reducing the necessity of extensive post-elution purification.

Unexpectedly, it has been discovered that the concentration of sugar in the elution buffer employed in an affinity membrane purification of glycosylated

proteins can be about half (or less) that of the elution buffer that provides equivalent eluting power in a conventional column affinity process, using, for example a Sepharose™ support matrix.

In the present elution method, for example, the protein can be eluted by contacting the membrane-bound ligand-ligate pair with an eluent having a sugar concentration of from about 0.05 M to about 0.4 M, and a salt concentration of from about 0.3 M to about 1.0 M. In addition, salt alone at the above concentration range can be used to elute glycosylated proteins. Salt solutions alone cannot be used at all to elute glycosylated proteins from lectin ligands immobilized on a non-membrane support matrix. This is illustrated in Table II.

Ion Exchange Chromatography

In addition to the steps enumerated above, the sample containing the glycosylated protein of interest can optionally be pre-treated, or post-treated, by ion-exchange chromatography. It has been found that greater purity of the protein product can sometimes be obtained when the sample is first treated by contacting it with an ion-exchange resin. In another embodiment of this step, the protein can be contacted with an ion exchange resin after its elution from the lentil lectin column. Both pre- and post-treatment by ion-exchange chromatography results in greater purity of the final product. For example, therapeutic grade IgM can be obtained by first separating the IgM using a lectin- activated affinity membrane, then running the IgM obtained from the lectin purification step through a column containing an anion exchange resin. The term "therapeutic grade" means that the glycosylated protein contains no detectable impurities.

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. EXAMPLES

Example 1

The ligate purified in this example was macrophage colony stimulating factor or MCSF, a glycoprotein; this was accomplished using immobilized lentil lectin (LL) from lens culinaris (Sigma Chemical Co., St. Louis, MO) as the affinity ligand and methyl-alpha-D-glucopyranoside (σMG) as a specific eluent. In conventional processes for the affinity purification of MCSF, the lentil lectin ligand is bound to Sepharose^TM gel supports, which exhibit dynamic capacities of about 0.8 mg/mL of bed. In the affinity membrane purification process of the present invention, the lentil lectin was immobilized on 1.5 mL

(0.5 mL membrane matrix volume or MMV) and 30 mL (10 mL MMV) hollow-fiber affinity membrane modules using FMP linking chemistry as described above. Approximately 1 to

2 mg of lentil lectin were immobilized per mL of membrane matrix Volume, leading to dynamic capacities for MCSF uptake in the range of 3 to 4 mg/mL MV and static or equilibrium capacities of about 9 to 10 mg/mL.

The lentil lectin membrane modules were loaded with a solution of MCSF containing 0.02 M Tris buffer, 0.1 M

NaCl, and 0.1% Tween at pH 7.4. Loading conditions and flowrates were typically chosen to provide good capture efficiencies for MCSF. For instance, in one experiment, 400 mL of MCSF-containing solution were processed in four cycles, each processing 100 mL at a filtrate flowrate of 2.0 mL/min. After loading, the membrane matrix was washed with a buffer containing only 0.02 M Tris and 0.1% Tween at pH 7.4. The composition of the solutions employed in the subsequent elution steps was varied in a series of experiments in an attempt to determine how low a

concentration of the αMG displacing sugar would prove effective in eluting MCSF from the affinity membrane matrix. In these experiments, the affinity membrane module was first loaded with MCSF to saturation (about 9 to 10 mg/mL MV). In all cases, the elution buffers contained 0.5 M NaCl, 0.02 M Tris and 0.1% Tween at pH 7.4. However, the concentration of αMG in the elution solution was varied from 0.0 to 0.4 M as shown in Table III. It can be seen that αMG concentrations significantly less than 0.4 M sufficed to displace a high fraction of the MCSF from the lentil-lectin affinity membrane. Concentrations of αMG of about 0.05 to 0.10 M are preferred in a practical affinity membrane process for MCSF purification.

TABLE III

EFFECT OF αMG CONCENTRATION ON ELUTION OF MCSF FROM LENTIL LECTIN AFFINITY MEMBRANE

*Elution buffers also contain 0.5 M NaCl, 0.02 M Tris, and 0.1% Tween at pH 7.4 In view of the reported increase in the rate of lectin leaching associated with the use of high concentrations of displacing sugars in the elution step of similar affinity purifications (Walzel, H. et al., (1989) Biomed. Biochim. Acta., 4:221) it is reasonable to believe that the lower αMG concentration which proved satisfactory in the affinity membrane purification process should lead to a longer useful lifetime of the membrane-immobilized lentil lectin ligand.

Table II sets out the results of a purification of

MCSF using an immobilized lentil lectin affinity membrane compared to lentil lectin immobilized on Sepharose™. As shown in Table II, MCSF purified using the lentil lectin affinity membrane was higher purity (85% by weight) than that obtained using the Sepharose™ support (70%).

Figures 2A and B illustrate the results of affinity purification of MCSF using lentil lectin membranes.

Figure 2A is a chromatogram showing the results of a GPC analysis of a cell culture supernatant from CHO cells containing recombinant MCSF which was produced by the cells. The cell culture sample has several broad peaks representing impurities in the sample. Figure 2B is a chromatogram showing the results of GPC analysis after lentil lectin affinity membrane purification. The results show a single peak of pure MCSF.

Example 2

Immobilized lentil lectin on Sepharose™ 4B was obtained from Sigma Chemical Co. (St. Louis, MO). A total of 1.0 ml of the lectin matrix was allowed to settle in a 4ml disposable column. The matrix was then washed with PBS (pH 7.4). The capacity of the Sepharose lentil lectin to bind a glycoprotein was assessed using Porcine Thyroglobulin obtained from Sigma Chemical Co. A thyroglobulin

solution of 1.0 mg/ml was allowed to flow over the

Sepharose™ matrix at a rate of 0.5 ml/min using gravity flow. After 25 mis had been processed, the Sepharose™ matrix was washed free of excess nonbound proteins with 25 mis of load buffer. The bound glycoprotein was then eluted with the following buffer: 20 mM Tris, 0.1% Tween 20, 0.5M NaCl, 0.2M αMG, pH 7.4. Glycoproteins eluted with each buffer were quantitated by measurement of optical density at 280 nm, assuming an extinction

coefficient, for a 1% solution of 14.0. The static

capacity for thyroglobulin uptake so determined was 3-4 mg/ml.

The capacity of the Sepharose™ lentil lectin to bind IgM was assessed using a commercially available purified IgM. An IgM solution of 1.0 mg/ml prepared in loading buffer was allowed to flow over the Sepharose matrix at a rate of 0.5 ml/min using gravity flow. After 25 mis had been processed, the Sepharose matrix was washed free of excess nonbound IgM with 25 mis of load buffer. The bound IgM was then eluted with the following buffer: 20 mM Tris, 0.1% Tween 20, 0.5M NaCl, 0.2M αMG, pH 7.4. IgM eluted with each buffer was quantitated by O.D. 280 analysis as described above. As shown in Table I, the static capacity for IgM uptake so determined was 1.0 mg/mL.

Poly (ethersulfone) -based affinity membrane hollow fibers were produced as described above (0.5 ml mv). The membrane fibers were activated with FMP during manufacture, as described in copending application USSN 07/258,406, which is incorporated herein by reference.

Lentil lectin was immobilized on the FMP-activated membrane using the following method:

1) The module was washed with loading buffer

(0.15M NaCl, 0.5% Tween 20, pH 7.8);

2) A lentil lectin (Iens culinaris) solution at 1.0 mg/ml in loading buffer was allowed to flow over the module at room temperature for 16-20 hours;

3) Excess loading solution was drained from the module;

4) Remaining activated groups were capped by

allowing a solution of 1.0M ethanolamine, pH 8.0 flow over the module at room temperature for 16-20 hours; and

5) The resulting lentil lectin activated module was stored in PBS, pH 7.4, 0.1% Thymerosal at 4°C. The static capacity of the lentil lectin module as assessed using Porcine Thyroglobulin was determined to be 12-15 mg/mL using the experimental protocol described in Example 1 above.

The capacity of the membrane-bound lentil lectin to bind IgM was assessed using a commercially available purified IgM. An IgM solution of 1.0 mg/ml prepared in loading buffer was allowed to flow over the membrane matrix at a rate of 2.0 ml/min using controlled pumps on a Sepracor Affinity-15 system (Sepracor, Inc.,

Marlborough, MA). After.25 mis had been processed, the membrane lentil lectin matrix was washed free of excess nonbound IgM with 25 mis of load buffer. The bound IgM was then eluted with the following buffer: 20 mM Tris, 0.1% Tween 20, 0.5M NaCl, 0.2M Mg , pH 7.4.

IgM eluted with each buffer was quantitated by O.D. 280 analysis as described above. Static capacity for IgM uptake was found to 3-4 mg/mL as shown in Table I.

Figures 1A and 1B illustrate the results of affinity purification of IgM using lentil lectin membranes.

Figure 1A is a chromatogram showing the results of a gel permeation chromatography (GPC) analysis of a mouse ascites fluid containing IgM before purification. The chromatogram shows several components besides the-IgM, which is represented by peak 1. Figure 1B shows the results of GPC analysis after lentil lectin-affinity membrane purification. The results show a single peak of pure IgM.

The purity of IgM obtained by lentil lectin affinity chromatography is significantly greater than that obtainable using lentil lectin immobilized on non-membrane supports, such as Sepharose™, as shown in Table IV:

TABLE IV

*The chromatogram peaks refer to Figure 4A and 4B, which show the level of purity of IgM purified using a lentil lectin ligand immobilized on an affinity membrane (Figure 4A) and Sepharose™ (Figure 4B). Peak 2 shown as PK2 in these Figures and in Table IV is an unknown protein contaminant. Albumin, shown as ALB PK, is the major contaminant of IgM preparations. As shown above, the presence of albumin and the other protein contaminants is minimized using the affinity membrane process.

Example 3

IgM was purified from mouse ascites fluid using immobilized lentil lectin from lens culinaris prepared as described in Example 2 above, as the affinity ligand and αMG as the specific eluent.

The lentil lectin membrane module was loaded with the ascites sample containing IgM. Loading conditions and flowrates were chosen to provide good capture

efficiencies for IgM. After loading, the membrane matrix was washed with a buffer containing 0.02 M Tris, 0.05% Tween 20, pH 7.4, to remove all non-bound proteins.

The bound IgM was removed by contacting the module with an elution buffer containing 0.02 M Tris, 0.05% Tween 20, pH 7.4, 0.5 M NaCl and 0.2 M αMG.

The results are shown in Figures 3A and 3B. Figure 3A is a GPC analysis of the mouse ascites sample before purification by lentil lectin, and Figure 3B shows the purified IgM obtained after lentil lectin purification.

Example 4

A mouse ascites sample (4ml) containing IgM was purified by ion exchange chromatography after

purification using the lentil lectin membrane process described in Example 3. In this step, the ascites sample was diluted 1:1 with an equal volume (4ml) of equilibration buffer (10mM sodium acetate, pH 4.5). The pH of the mixture was adjusted to 4.5 with concentrated acetic acid. The mixture was loaded onto a 10 ml bed volume column containing an SP Trisacryl Plus M ion exchange support (IBF Biotechnics) at 0.5 ml/min.

The column was washed to baseline with the equilibration buffer, then with 2-3 column volumes of wash buffer 1 (25 mM phosphate, pH 6.0), until the baseline was obtained. The column was then washed with 4-5 column volumes of wash buffer 2 (PBS, pH 6.0). The eluant buffer (PBS, 0.25M sodium chloride (NaCl), pH 6.0) was then passed through the column in the amount of 2-3 column volumes, and the fractions containing IgM were collected. Material remaining on the column was removed by washing the column with a strip buffer (1M NaCl, 0.02% sodium azide). The results are shown in Figure 3C.

Example 5

Activated affinity membranes were prepared using glutaraldehyde linking chemistry. In this method, polyethersulfone-based hollow fibers were prepared according to the method described in co-pending patent application USSN 07/258,406. The fibers were washed in deionized water and autoclaved for 15 minutes at 120ºC. The fibers were then washed with 2L acetonitrile for 30 minutes, and with the deionized water wash for 30

minutes.

The fibers were activated by exposure to 10%

ethylene glycol diglycidyl ether (EDGDE) and 0.6N sodium hydroxide (NaOH) according to the following procedure : the fibers were heated to 80 º C , and 3L of de ionize d water was added. To the stirring mixture, 40g of 50% NaOH was added, and allowed to mix for 10 minutes. The mixture was added to a larger vessel, and 1.45 L of deionized water and 500 ml EGDGE were added and allowed to circulate for three hours. The fibers were then rinsed with deionized water.

The activated fibers are then coated with

polyethyleneimine (PEI) according to the following procedure. Water was added to the vessel containing the fibers. To the stirring solution, 240g of 0.6 N NaOH solution and 333.g of PEI solution (30% in water) were added. 2L of deionized water was added to the mixture. The fibers and the PEI mixture then heated to 80°C and maintained for 4 hours. The fibers were then rinsed with water for 15 minutes.

The fibers were then activated with glutaraldehyde according to the following procedure: 5L of phosphate buffered saline (PBS) was added to 500 g of 25% glutaraldehyde in water and 15.7 g of NaCNBH₄, and the mixture was stirred until the NaCNBH₄ dissolved. The fibers and the solution were placed in a coating apparatus and maintained for 4 hours at room temperature. The fibers were then rinsed with water and coated with PEI as described above. The glutaraldehyde activation step was repeated, except that the coating was maintained for two hours rather than four. The fibers were then dried at 80ºC overnight.

Lentil lectin was immobilized on the glutaraldehyde membrane in the following manner:

1) The module was washed with loading buffer

(0.15M NaCl, 0.05% Tween 20, pH 7.0);

2) A lentil lectin (lens culinaris) solution at 1.0 mg/ml in loading buffer was allowed to flow over the module at room temperature for 16-20 hours; 3) Excess loading solution was drained from the module;

4) Remaining activated groups were capped and the protein bonds stabilized by allowing a solution of 1.0 M ethanolamine, pH 7.0, 0.01M sodium cyanoborohdride, to flow over the module at room temperature for a period of two hours;

5) Excess capping solution was drained from the module; and

6) The resulting lentil lectin module was stored in PBS, pH 7.4, 0.1% Thymerosal at 4°C.

Example 6

Lentil lectin immobilized on the affinity

membrane was used to purify murine IgM contained in ascites fluid harvested from a donor mouse. A

typical purification of the ascites fluid IgM using the lentil lectin affinity membranes involved a

dilution of the fluid 1:5 or 1:10 with 50 mM Tris buffered saline. Following a 0.22 um filtration the IgM solution was processed with the lentil lectin affinity membrane as described previously.

Analysis of the recovered product included SDS polyacrylamide gels (PAGE) run under reducing

conditions, ELISA quantitation of IgM and Western blotting onto nitrocellulose membranes and

identification of the IgM heavy chain with a murine IgM heavy chain specific antibody probe. Samples analyzed were the intial feed s tream , the filtrate or processed feed stream and the eluted IgM product.

SDS PAGE stained with Comassie Blue stain

revealed that the feed stream contained large amounts of murine albumin, as well as other

unidentified proteins typical of serum. Western blotting and immunostaining for the IgM heavy chain revealed a large staining band corresponding to the appropriate molecular weight for IgM heavy chain.

On SDS PAGE the processed feed stream,

filtrate, was similar in number and quantity of Comassie Blue staining bands as the beginning feed stream. However, the Western blot with immuno- staining for IgM revealed that the IgM had been depleted when processed by the lentil lectin

affinity fibers.

The SDS PAGE analysis of the eluted proteins showed that there was a significant decrease in the number of Commasie Blue staining bands, with the only major band being consistent with the albumin band. The albumin content had been significantly (more than 90%) reduced. The Western Blot with immunostaining revealed a very heavy staining band corresponding to the IgM heavy chain. The inability to reliably stain the IgM heavy chain on SDS PAGE with Commasie Blue was consistent with standards of purified IgM obtained from commercial suppliers.

ELISA data on all the above SDS PAGE samples revealed that the lentil lectin affinity fibers were capturing more than 95% of the IgM offered them in the feed stream. The final product purity as determined by the ratio of total protein, determined by O.D. 280, E^1%=.14, to total IgM, determined by ELISA, was approximately 70-80%.

Claims

CLAI MS

1. A method for producing immobilized-lectin affinity membranes, comprising the steps of:

a) contacting a microporous polymer membrane with at least one activating reagent under conditions sufficient to cause activation of the membrane surface by incorporation of functional groups thereon; and

b) contacting the membrane in step (a) with the lectin under conditions sufficient for covalent attachment of the lectin to the activated membrane surface through the functional groups to occur.

2. A method of Claim 1 wherein the microporous polymer membrane comprises poly (ethersulfone) or

poly(ethersulfone)/polyethylene oxide).

3. A method of Claim 2 comprising the additional step of coating the microporous polymer membrane with hydroxyethyl cellulose. 4. A method of Claim 2 comprising the additional step of coating the microporous polymer membrane with polyethyleneimine.

5. A method of Claim 3 wherein the activating reagent is FMP.

6. A method of Claim 4 wherein the activating reagent is glutaraldehyde.

7. A method of Claim 1 wherein the static capacity of the membrane for IgM is at least 2 mg/ml. 8. A method of Claim 1 wherein the static capacity of the membrane for MCSF is at least 5 mg/ml.

A method for eluting a glycosylated protein which is associated with an immobilized lectin bound to a microporous membrane, from said lectin, comprising the steps of: contacting the microporous membrane with an eluent that contains sugar having a concentration of from about 0.05 M to about 0.4 M and a salt having a concentration of from about 0.3M to about 1M under conditions such that the glycosylated protein is dissociated from the lectin.

10. A method of Claim 9 wherein the glycosylated protein is MCSF.

11. A method of Claim 10 wherein the MCSF is greater than 80% pure. 12. A method of Claim 9 wherein the glycosylated protein is IgM.

13. A method of Claim 12 wherein the IgM is greater than 80% pure.

14. A method of Claim 12. wherein the IgM contains less than 10% albumin contaminant.

15. A method of Claim 9 wherein the sugar is methyl-α-D- glucopyranoside or methyl-α-D-mannopyranoside. 16. A method of Claim 9 wherein the concentration of the sugar is from about 0.05 M to about 0.2 M.

17. A method of Claim 9 wherein the salt is about 0.5 M sodium chloride.

18. A method of Claim 9 further comprising the step of contacting the glycosylated protein with an ion exchange resin under conditions sufficient to further purify the glycosylated protein.

19. A method of Claim 18 wherein the glycosylated

protein is contacted with the ion exchange resin after binding to the lectin.

20. A method of Claim 18 wherein the ion exchange resin is an anion exchange resin.

21. A method of eluting a glycosylated protein from a lectin bound to a microporous membrane comprising the steps of: contacting the microporous membrane with a eluent having a sugar concentration of from about 0.5 M to about 0.2 M and a salt concentration of about 0.5M under conditions sufficient to

dissociate the glycosylated protein from the lectin.

22. A method of Claim 21 wherein the glycosylated protein is MCSF.

23. A method of Claim 21 wherein the glycosylated

protein is Immunoglobulin M. 24. A method of Claim 21 wherein the lectin is lentil lectin.

25. A method of Claim 21 wherein the eluent is an

aqueous salt solution having a salt concentration of about 0.5 M. 26. A method of Claim 21 wherein the sugar is methyl-α- D-glucopyranoside or methyl-α-D-mannopyranoside.

27. An affinity membrane having lectin covalently bound thereto made by the method of Claim 1.

28. An affinity membrane having lectin covalently bound thereto made by the method of Claim 5.

29. An affinity membrane having lectin covalently bound thereto made by the method of Claim 6.

30. A lectin-bound affinity membrane comprising:

a) a microporous polymer membrane coated with a coating material having functional groups;

b) lectin molecules; and c) a linker moiety covalently binding the lectin molecules to the functional groups of the coating.

31. A lectin-bound affinity membrane of Claim 30 wherein the coating material is hydroxyethylcellulose and the linker moiety is FMP.

32. A lectin-bound affinity membrane of Claim 30 wherein the coating material is polyethyleneimine and the linker moiety is glutaraldehyde. 33. A lectin-bound affinity membrane of Claim 30 wherein the hydrophobic polymer is selected from the group consisting of: polysulfone, poly(ethersulfone) and poly(ethersulfone)/poly(ethylene oxide).

34. A lectin-bound affinity membrane of Claim 30 wherein the lectin is lentil lectin.